Plectin 1f scaffolding at the sarcolemma of dystrophic (mdx) muscle fi bers through multiple interactions with β-dystroglycan

Günther A. Rezniczek

  THE JOURNAL OF CELL BIOLOGY

  © The Rockefeller University Press $15.00 The Journal of Cell Biology, Vol. 176, No. 7, March 26, 2007 965–977 http://www.jcb.org/cgi/doi/10.1083/jcb.200604179 JCB 965

  Published March 26, 2007 Supplemental Material can be found at:

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  The online version of this article contains supplemental material.

  Correspondence to Gerhard Wiche: gerhard.wiche@univie.ac.at Abbreviations used in this paper: ABD, actin-binding domain; βDG, β-dystroglycan; DGC, dystrophin–glycoprotein complex; DMD, Duchenne MD; EDL, extensor digitorum longus; IB, immunoblotting; IF, intermediate fi lament; IFM, immuno- fl uorescence microscopy; IP, immunoprecipitation; MD, muscular dystrophy; MyHC, myosin heavy chain.

  Coimmunoprecipitation and in vitro binding assays using recombinant protein fragments revealed the direct bind- ing of plectin to dystrophin (utrophin) and β-dystroglycan, the key components of the dystrophin–glycoprotein complex. We propose a model in which plectin acts as a universal mediator of desmin intermediate fi lament anchorage at the sarcolemma and Z-disks. It also explains the plectin phenotype observed in dystrophic skeletal mus- cle of mdx mice and Duchenne muscular dystrophy patients.

  n skeletal muscle, the cytolinker plectin is prominently expressed at Z-disks and the sarcolemma. Alternative splicing of plectin transcripts gives rise to more than eight protein isoforms differing only in small N-terminal sequences (5–180 residues), four of which (plectins 1, 1b, 1d, and 1f) are found at substantial levels in muscle tissue. Using plectin isoform–specifi c antibodies and isoform ex- pression constructs, we show the differential regulation of plectin isoforms during myotube differentiation and their localization to different compartments of muscle fi bers, identifying plectins 1 and 1f as sarcolemma-associated isoforms, whereas plectin 1d localizes exclusively to Z-disks.

  1 1 Max F. Perutz Laboratories, Department of Molecular Cell Biology, University of Vienna, A-1030 Vienna, Austria 2 Department of Physiology, Anatomy, and Genetics, Medical Reasearch Council Functional Genetics Unit, Oxford OX1 3QX, England, UK 3 Centre for Developmental and Biomedical Genetics, Department of Biomedical Science, University of Sheffi eld, Western Bank, Sheffi eld S10 2TN, England, UK

  

3

and Gerhard Wiche

  2 Steve J. Winder,

  1 Kay E. Davies,

  1 Christina Abrahamsberg,

  1 Doris Schneller,

  1 Siegfried Reipert,

  1 Branislav Nikolic,

  1 Patryk Konieczny,

  Plectin 1f scaffolding at the sarcolemma of dystrophic (mdx) muscle fi bers through multiple interactions with β-dystroglycan

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Introduction

  Muscular dystrophies (MDs) are a group of clinically and genetically heterogeneous diseases characterized by progres- sive muscle wasting. Lack of dystrophin leads to the most com- mon form, Duchenne MD (DMD), but MD can also result from mutations in genes whose products are not known to associate with the DGC (Burton and Davies, 2002). Most patients with plectin defects, who mainly suffer from various subtypes of the skin blistering disease epidermolysis bullosa (Pfendner et al., 2005), have also been diagnosed with MD, and muscle phenotypes have been observed in plectin-defi cient mice (Andrä et al., 1997).

  Components of the DGC are part of the costameric protein network that, among other proteins, also includes integrins, vinculin, talin, α-actinin, and caveolin-3. Costameres are sub- sarcolemmal protein assemblies that circumferentially align in register with the Z-disks of peripheral myofi brils (for reviews see Spence et al., 2002; Ervasti, 2003); some authors include elements located above M-lines and in longitudinal lines in this term (Bloch et al., 2002).

  Transmission of force from skeletal muscle myofi brils to the ECM is thought to be mediated largely by intermediate fi la- ments (IFs). Several IF proteins are expressed in muscle, in- cluding vimentin, nestin, synemin, syncoilin, lamins, cytokeratins, and desmin, the major muscle-specifi c IF protein (for review see Paulin and Li, 2004). The desmin IF network forms a 3D scaffold surrounding Z-disks, extends from one Z-disk to the next, and fi nally connects the contractile apparatus to the plasma membrane at the level of Z-disks but also to organ- elles such as mitochondria and the nucleus (for review see Capetanaki, 2002). The dystrophin–glycoprotein complex (DGC) has been implicated in mediating the IF-ECM link through syn- coilin and synemin, which interact with desmin and bind to the DGC protein α-dystrobrevin (Bellin et al., 2001; Newey et al., 2001; Poon et al., 2002). The DGC is a large protein complex consisting of integral membrane proteins (α- and β-dystro- glycan [βDG], α-, β-, γ-, and δ-sarcoglycan, and sarcospan), the >425-kD large actin-binding protein dystrophin, and dystrophin- associated proteins such as the syntrophins and α-dystrobrevin.

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  The cytolinker protein plectin is prominently expressed in stri- ated muscle cells and has been visualized at Z-disks, the sarco- lemma, and at mitochondria (Wiche et al., 1983; Schröder et al., 1997; Reipert et al., 1999; Hijikata et al., 2003), but the mole- cular mechanisms involved in plectin-related muscle disease/ defects are unknown.

  Plectin is a large (M r > 500,000) protein consisting of N- and C-terminal globular domains separated by an 200-nm– long rod. The N-terminal domain contains a multifunctional actin-binding domain (ABD; Andrä et al., 1998) that is capable of also interacting with integrin β4 (Rezniczek et al., 1998; Geerts et al., 1999) and vimentin (Sevcik et al., 2004) and also contains binding sites for nesprin-3 (Wilhelmsen et al., 2005) and the nonreceptor tyrosine kinase Fer (Lunter and Wiche, 2002). The C-terminal domain contains binding sites for IFs (Nikolic et al., 1996), the γ1 subunit of AMP kinase (Gregor et al., 2006), and the PKC scaffolding protein RACK1 (Osmanagic-Myers and Wiche, 2004). Several different plectin isoforms, which are generated by tissue and cell type–dependent alternative splicing of transcripts from a single gene with >40 exons, form the basis for its broad versatility (Fuchs et al., 1999; Rezniczek et al., 2003). Isoforms with eight alternative N termini have been identifi ed, and specifi c functions have been linked to distinct isoforms. Plectin 1a anchors keratin IFs to hemidesmosomes in basal keratinocytes (Andrä et al., 2003), and a specifi c role in fi broblast and T cell migration has been demonstrated for plectin 1 (Abrahamsberg et al., 2005).

  In skeletal muscle, four isoforms (plectins 1, 1b, 1d, and 1f) are expressed at considerable levels. In this study, we address the following issues: Where on the subcellular level are these plectin isoforms localized in muscle fi bers? What are their muscle-specifi c (novel) binding partners? Are they differen- tially regulated during differentiation? What role do they play in dystrophic muscle, such as that of mdx mice?

  Plectins 1d, 1f, 1b, and 1, the isoforms most abundantly expressed in skeletal muscle, show relative mRNA ratios of >10:4:3:1, respectively (Fuchs et al., 1999). To obtain data about their expression and localization in skeletal muscle on the protein level, we isolated the quadriceps, a typical fast-twitch muscle composed of mainly type 2 fi bers, from 10-wk-old mice and processed it for immunolabeling. Anti–pan-plectin antiserum revealed strong subsarcolemmal and moderate sarcoplasmic staining in cross sections of small diameter fi bers and only faint sarcoplasmic and sarcolemmal staining in larger diameter fi bers (Fig. 1 C). On longitudinal sections, Z-disks were stained in all fi bers, but the signal was much stronger in small diameter fi bers, where additionally the plasma membrane was stained (Fig. 1 A). These fi bers, which showed strong autofl uorescence at 488 nm (Fig. 1, F and H; insets), were positive for myosin heavy chain (MyHC)–2A (Fig. 1 B; also see E, G, and I), whereas those with larger diameters were MyHC-2B positive (Fig. 1 K). Therefore, it appears that in quadriceps, fast 2A fi bers express plectin at higher levels than type 2B fi bers, as has previously been reported for type 2 compared with slow type 1 fi bers (Schröder et al., 1997). Double immunolabeling of plectin 1f and MyHC-2A on longitudinal sections revealed this plectin iso- form to be located at Z-disks in 2A fi bers but to be hardly ex- pressed in 2B fi bers (Fig. 1, D and E; and not depicted). On cross sections, 2A fi bers showed moderate sarcoplasmic plectin 1f– specifi c staining as well as irregular and weak staining of the membrane (Fig. 1, F and G). Staining of longitudinal sections using a plectin 1–specifi c antiserum revealed this isoform to be much less abundant, if at all present, at Z-disks. However, a strong

  Figure 1. Immunolocalization of plectin isoforms in quadri- ceps of 10-wk-old mice. Consecutive (except A–C) serial lon- gitudinal and cross sections were stained with antibodies (pan-plectin) recognizing all plectin isoforms (A and C) or spe- cifi cally isoforms 1f (D and F) or 1 (H and J); in addition, anti- bodies specifi c to MyHC-2A (B, E, G, and I) and -2B (K), all in combination with Cy5-labeled secondary antibodies, were used. The boxed area in D (bottom right) is shown enlarged in the inset. The insets in F and H show autofl uorescence after excitation at 488 nm recorded in the standard FITC channel. Bar, 50 μm.

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Results Muscle fi ber type–dependent expression and isoform-specifi c subcellular localization of plectin

  signal came from sarcolemma-associated structures, primarily in 2A fi bers (Fig. 1 H). On cross sections, plectin 1–specifi c signals were detected as irregularly distributed accumulations at the sarcolemma of 2A but not 2B fi bers (Fig. 1 J).

  As we were unsuccessful in generating isoform-specifi c antibodies directed against plectins 1b and 1d, we ectopically expressed and visualized GFP fusions of all four full-length plectin isoforms (1, 1b, 1d, and 1f) in myotubes (Fig. 2, A–D). Plectin 1 was expressed in a diffuse dotty pattern throughout the cytosol (Fig. 2 A; see virtual cross sections in insets 1 and 2) and was concentrated in the vicinity of nuclei. Immunolabeling with antibodies specifi c for sarcomeric α-actinin, a marker for Z-disks, revealed that areas positive for α-actinin were com- pletely devoid of plectin 1 (Fig. 2 A, a and b; see areas marked by identically positioned arrowheads). Plectin 1b was distrib- uted throughout the sarcoplasm in a pattern somewhat more patchy but similar to that observed for plectin 1, also mostly ex- cluding areas that were positive for α-actinin (Fig. 2 B; a, b, and cross sections). Plectin 1d was located exclusively at structures identifi ed as Z-disks (Fig. 2 C; arrowheads in a and b indicate the same exemplary positions). Contrary to expectations based on the immunostaining of tissue sections, plectin 1f was found not to be associated with Z-disks (Fig. 2 D). However, the ob- served sarcolemma association of this isoform was impressively confi rmed (Fig. 2 D, virtual cross sections 1–4; a and b show individual confocal sections as indicated in panel 1). Immuno- labeling of in vitro–differentiated C2C12 cells with plectin 1– and 1f–specifi c antibodies revealed the same localization of the native isoforms (unpublished data).

  To further investigate the sarcolemma association of plectin, extensor digitorum longus (EDL) muscle was teased into sin gle fi bers, which were immunolabeled for plectin and βDG, a costameric membrane marker (Fig. 2 E). Both proteins co- localized in costameric structures. Whereas βDG staining resembled a gridlike pattern (Z-disks and longitudinal lines), pan-plectin serum revealed prominent Z-disk and perinuclear localization and only a rare association of plectin with longitu- dinal lines. Virtual cross and longitudinal sections (Fig. 2 E, insets 1 and 2) through confocal stacks showed plectin at the sarcolemma but also extending into the fi bers in regular intervals. Analysis with isoform-specifi c antibodies showed distinct stain- ing patterns for plectins 1 and 1f. Whereas plectin 1 was found in the perinuclear area, at longitudinal lines, and in a dotty pattern at Z-disks (Fig. 2 F), plectin 1f was strongly expressed at

  Figure 2. Expression of full-length plectin isoforms in differentiated myotubes and immunostaining of teased muscle fi bers. (A–D) Full-length plectin 1 (A), 1b (B), 1d (C), or 1f (D) ex- pression plasmids (with C-terminal EGFP; green) were trans- fected into myoblasts 12–16 h before differentiation. After 96 h, differentiated myotubes were fi xed with methanol and processed for immunolabeling using antibodies specifi c for sarcomeric α-actinin (A–C) or caveolin-3 (D). Secondary antibodies were Texas red labeled (red), and nuclei were visualized with Hoechst 33342 (blue). Main panels show composites of confocal stacks; dotted frames indicate areas shown in more detail in panels a and b of A–D. Numbered lines indicate confocal sections shown in correspondingly labeled insets; virtual cross sections were reconstructed from confocal stacks using LSM imaging software. Horizontal lines in insets 1–4 of D indicate the positions of the planes shown in panels a and b of D. Arrowheads in A and C indicate identi- cal exemplary positions in panels a and b. (E–H) Teased fi bers were prepared and processed for IFM as described in Materials and methods. Primary antibodies used were anti- serum #123 to plectin (E), anti–plectin 1 (F), anti–plectin 1f (G and H), monoclonal anti-βDG (E), anti–α-actinin (F and G), and antidystrophin (H). Plectin-specifi c antibodies were de- tected with Cy3-conjugated secondary antibodies (green), and all others were detected with Cy5-conjugated secondary antibodies (red). Panels a and b show individual colors of the merged images. The virtual sections shown in insets 1 and 2 in E are from a different fi ber. The line in H indicates the plane of section shown in inset 1. Bars in A and E apply to A–D and E–H, respectively; bars in panel b of C and E apply to panels a and b of A–D and E–H, respectively. Bars, 20 μm.

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  Z-disks and tightly encircled nuclei (Fig. 2 G). Intriguingly, when the staining patterns for both isoforms are merged, they match that observed with pan-specifi c plectin antibodies, suggesting that plectins 1 and 1f are the major sarcolemma-associated isoforms. Costaining of plectin 1f with dystrophin revealed a partial colocalization of both proteins (Fig. 2 H). At the sarco- lemma, plectin 1f was concentrated at Z-disks and extended into the fi ber, whereas dystrophin was limited to the sarcolemma but was also found between Z-disks (Fig. 2 H, inset 1).

  Coexpression of plectin 1f and dystrophin during myoblast differentiation

  To defi ne the role of plectin in the process of differentiation from myoblasts to myotubes, we profi led the expression of plectin and other skeletal muscle proteins (Fig. 3). After 96 h of differentiation, myoblast cultures had formed myotubes that started to twitch (unpublished data). During differentiation, plectin isoform 1 expression peaked at 8–16 h, which is similar to that of utrophin. Plectin 1f was expressed only later, starting between 24 and 48 h, and reached plateau levels after 72 h. Interestingly, dystrophin showed a very similar expression pro- fi le, whereas βDG was detectable earlier (16 h), and caveolin-3 was not detected before 48 h. Integrin α7B was already ex- pressed in myoblasts and showed peak levels after 48 h of differentiation.

  Plectin interacts with the EF-ZZ domains of dystrophin and utrophin

  The spatially and temporally coordinated expression of plec- tin 1f and dystrophin suggested a possible direct interaction of both proteins. In immunoprecipitation (IP) experiments using

  IP lysates (see Materials and methods) from wild-type muscle tissue, plectin coprecipitated with dystrophin and vice versa (Fig. 4 A, lanes 5 and 7). From mdx IP lysates, which were used as negative controls, plectin was not precipitated using dystrophin antibodies (Fig. 4 A, lanes 6 and 8). Interestingly, although plectin was expressed at higher levels in mdx com- pared with wild-type muscles (Fig. 4 A, lanes 1 and 2), less of it was immunoprecipitated from mdx (Fig. 4 A, lanes 5 and 6; also see Fig. 6, A and C), indicating a shift of plectin into an insoluble pool. Utrophin and plectin were coprecipitated from rat fibroblast lysates (unpublished data). To identify inter- acting subdomains of the proteins, we immobilized His-tagged fragments of utrophin, including its N-terminal ABD, the C ter- minus, and the entire WW-ZZ domain as well as its three sub- domains WW, EF, and ZZ on nitrocellulose membranes and overlaid them with various plectin samples (Fig. S1 A, avail- able at http://www.jcb.org/cgi/content/full/jcb.200604179/DC1; summarized in Fig. 4 B). Using either purified full-length plectin or plectin-rich cell lysates, we found plectin bound to the ABD and WW-ZZ domain of utrophin but not to its C-terminal part. A recombinant plectin ABD showed similar specifi city, although its binding to the utrophin ABD was very weak compared with that of full-length plectin. When over- laid onto WW-ZZ subdomains, positive signals were obtained for the EF and ZZ domains, whereas a fragment encoded by exons 9–12 of plectin did not show binding to any of the utro- phin fragments used. Confi rming these results, a Eu 3+ -labeled version of the plectin ABD specifi cally bound to WW-ZZ domains of utrophin and dystrophin when overlaid onto micro- titer plate–immobilized proteins (Fig. S1 B). Furthermore, the WW-ZZ domain of dystrophin competed with that of utrophin for plectin ABD binding as did actin (with considerably higher effi ciency; Fig. S1 C). This suggested that simultaneous binding of actin and WW-ZZ domains to the plectin ABD was unlikely to occur.

  Up-regulation of sarcolemmal plectin in mdx muscle

  To defi ne the relation of plectin with costameric membrane com- plexes in muscles lacking dystrophin, we characterized plectin localization in skeletal muscle fi bers at different stages of MD in mdx mice by immunolabeling cross sections of quadriceps

  Figure 3. Protein expression profi ling during the differentiation of myo- blasts in vitro. Myoblasts isolated from plectin (+/+)/p53 (−/−) mice were differentiated in vitro for up to 96 h. Immediately before the start of differ- entiation and after 4, 8, 16, 24, 48, 72, and 96 h, cells were lysed in sample buffer, and proteins were separated by SDS-PAGE on 5% (plectin, dystrophin, and utrophin) or 15% (βDG, caveolin-3, integrin α7B, and tubulin) gels and analyzed by IB. Bands were scanned and evaluated den- sit ometrically. Signals were normalized to tubulin and represent the means of at least triplicate experiments. 100% corresponds to the highest expres- sion of each protein during the course of differentiation. Error bars (SD; rarely >10%) have been omitted for clarity.

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  Published March 26, 2007 from 2-, 4-, and 14-wk-old animals with plectin-specifi c anti- bodies (Fig. 5, A–K). Compared with normal muscle, no differ- ences were observed at the (prenecrotic) age of 2 wk (not depicted) and in unaffected areas of quadriceps from 4-wk-old (peak necrotic) mdx mice (Fig. 5, A and B and D and E). Plectin 1f was found in the sarcoplasm and irregularly at the sarco- lemma, whereas plectin 1 was localized only in subsarcolemmal accumulations. Dystrophic areas were clearly distinguished by the presence of high numbers of small-diameter fi bers with cen- tralized nuclei and loose connective tissue, expressing very high levels of plectins 1 and 1f (Fig. 5, C and F). After 14 wk, most fi bers had already passed through one round of degeneration/ regeneration, and, as was expected from fi ndings in DMD muscles (Schröder et al., 1997), we observed increased plectin staining at the sarcolemma of regenerated fi bers when using a pan-plectin antibody (unpublished data). Isoform-specifi c anti- bodies revealed that this increased sarcolemmal staining was caused by the up-regulation of plectin 1f (Fig. 5, H and I vs. G). This was especially evident in 2B fi bers, which are identifi ed as large-diameter fi bers lacking autofl uorescence (Fig. 5 I; insets in G–I show autofl uorescence). For plectin 1, on the other hand, we could not detect notable differences between mdx and con- trol samples (Fig. 5, J and K). Thus, during the regeneration of mdx muscles, plectins 1 and 1f were both up-regulated in regenerating myotubes, but only plectin 1f associated with the sarcolemma and stayed there at high levels after regeneration was complete.

  To obtain quantitative estimates of plectin up-regulation in mdx mice compared with other sarcolemma-associated proteins, we prepared KCl-washed microsomes from skeletal muscle of 8–10-wk-old mdx and control mice (Ohlendieck and Campbell, 1991; Cluchague et al., 2004). Compared with total muscle lysates (Fig. 6 A), microsome fractions from control muscle were enriched in DGC components (dystrophin, utro- phin, and βDG) and the membrane markers caveolin-3 and integrin α7B; in addition, actin and plectins 1 and 1f but not tubulin were detected in microsomes (Fig. 6 B). Comparing corresponding control and mdx samples, we found increased levels of plectin ( 170%) and utrophin ( 140%) in total mus- cle lysates, whereas those of actin were similar (Fig. 6 C). Total plectin was two- to threefold more abundant in mdx versus con- trol microsome fractions, with relative levels of plectin 1 and plectin 1f of 300% and 150%, respectively. Interestingly, the levels of utrophin in the sarcolemmal fraction were only 50% of those found in the wild type, suggesting a weaker membrane association of utrophin in mdx muscle. With our lysis protocol (see Materials and methods), the levels of βDG were found at 50% compared with the wild type, although

  mdx βDG levels as high as 100% of wild type have been

  reported when samples were treated with cholate detergent (Cluchague et al., 2004). Caveolin-3 appeared slightly increased, and no notable difference was observed in the case of actin.

  Interestingly, the mdx levels of integrin α7B were approxi- mately fourfold increased (Fig. 6 D). Thus, these biochemical data were in agreement with the observed up-regulation of plec- tin in mdx muscle and the sarcolemma association of isoform 1f observed in the immunolabeling of tissue sections.

  Utrophin is likely not the preferred binding partner of plectin at the sarcolemma of mature mdx muscle fi bers

  To assess whether utrophin was substituting for dystrophin as a linker protein between βDG and plectin in dystrophin-defi cient muscle, we stained cross sections of mdx gastrocnemius for βDG and utrophin (Fig. 7, A–L). The antiserum to βDG gave a strong signal in control samples of all ages (2, 4, and 14 wk;

  Figure 4. Co-IP of plectin with dystrophin and direct binding of plectin to the utrophin/dystrophin EF-ZZ domain via its ABD. (A) Total and IP lysates of wild- type (wt) and mdx muscle tissues were prepared as described in Materials and methods. IP lysates were incubated without (IP: Control) or with antibodies spe- cifi c for plectin (IP: Ple) or dystrophin (IP: Dys). After separation and transfer to nitrocellulose, total lysates and precipitated samples were probed with anti- bodies specifi c for plectin (top) or dystrophin (bottom). (B) Schematic drawing of plectin and utrophin show- ing locations of binding interfaces, fragments used in blot overlay assays (bars above and below drawings), and a summary of binding data. Actual data are shown in Fig. S1 (available at http://www.jcb.org/ cgi/content/full/jcb.200604179/DC1). N and C termini of proteins are indicated. ABD, actin-binding domain. Overlaid plectin was either purifi ed from cell cultures (C6) or contained in cell lysates (804G). ++, strong binding; +/−, weak binding; −, no binding.

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  Fig. 7, A, E, and I) and reduced but still clearly positive sig- nals in the corresponding mdx samples (Fig. 7, C, G, and K). Actively regenerating mdx fi bers, which are marked by small diameters and centralized nuclei, showed a much stronger staining at the age of 4 wk that was almost similar in intensity to the sig- nal in control fi bers (Fig. 7 G). Utrophin was strongly expressed at the sarcolemmas of 2-wk-old control animals, whereas only limited staining was observed in corresponding mdx muscle samples (Fig. 7, compare B with D). At later developmental stages of normal muscle, utrophin was detectable at myotendi- nous (Fig. 7 F, arrowheads) and neuromuscular junctions (not depicted), but no general sarcolemmal staining was observed (Fig. 7, F and J). In the case of 4- and 14-wk-old mdx muscle, utrophin was present exclusively in regenerating small-diameter fi bers (Fig. 7, H and L; asterisks) and at myotendinous junctions (Fig. 7 H, arrowheads). Faint sarcolemmal utrophin-specifi c staining that was not visible in wild-type muscle was observed in mdx muscle (Fig. 7, H and L). The low levels of utrophin and the positive identifi cation of βDG at the sarcolemma of mdx muscle prompted us to costain teased wild-type and mdx muscle fi bers for plectin and βDG (Fig. 7, M–R). Both mAbs as well as an antiserum to βDG revealed that the gridlike staining pattern typical for costameres was lost in mdx fi bers, and, instead, βDG was found exclusively above Z-disks together with plectin 1f (Fig. 7, compare the insets of N and P, which are magnifi ed in Q and R). We also examined microsome fractions and sections prepared from muscles of mdx/utr

  −/−

  mice and found a similar situation as in mdx muscles (Fig. S2, available at http://www .jcb.org/cgi/content/full/jcb.200604179/DC1).

  Direct interaction of plectin with DG via multiple binding sites

  The redistribution of βDG to sites above Z-disks where plectin 1f was concentrated suggested that plectin could directly inter- act with the cytoplasmic domain of βDG. Co-IP of both pro- teins from lysates of C2C12 myoblasts, mouse keratinocytes, and the human colon adenocarcinoma cell line CaCo-2 using anti-βDG antibodies was successful (Fig. 8 A). Using no or irrelevant antibodies, neither plectin nor βDG was detectable in the corresponding precipitates (unpublished data). Plectin and βDG could also be coprecipitated from lysates of skeletal mus- cle from mdx mice (Fig. 8 B). Immuno-EM confi rmed the close association of both proteins at the sarcolemma of muscle fi bers (Fig. 8 C). When the plasma membrane was lost as a result of Triton X-100 extraction, βDG remained anchored to subsarco- lemmal fi lamentous structures (Fig. 8, white arrowheads), which were also positive for plectin (Fig. 8 D). βDG and plectin la beling was most prominent at subsarcolemmal regions over- lying Z-disks. Additionally, the plectin label was concentrated at the periphery of Z-disks (Fig. 8 E).

  To map the βDG-binding sites on plectin, a panel of His-tagged plectin fragments representing different structural domains (Fig. 9, A and B) were blotted onto nitrocellulose and overlaid with the cytoplasmic domain of βDG. The WW-ZZ domain of dystrophin, which binds to the C terminus of βDG (Ilsley et al., 2002), and the utrophin ABD or BSA were used as positive and negative controls, respectively. Using βDG- specifi c antibodies for detection, we found an interaction of βDG with two nonoverlapping plectin fragments (Fig. 9 C). One was en- coded by plectin exons 12–24, representing part of the plakin domain located C terminally of the ABD within the plectin N-terminal globular domain, and the other corresponded to the C terminus of plectin, starting within repeat domain 4. No other protein tested showed binding except for the dystrophin WW- ZZ domain. To narrow down the region of βDG involved in binding to plectin, a fragment (βDGcyt∆DBS) corresponding to roughly 70% of the βDG cytoplasmic domain (lacking the C-terminal region containing the dystrophin/utrophin-binding motif) was overlaid onto the same panel of recombinant pro- teins (Fig. 9 D). It bound to both plectin fragments identifi ed before but bound much weaker to the C-terminal plectin frag- ment (Ple R 4 -C), suggesting that additional C-terminal βDG sequences were needed for effi cient binding to this fragment.

  As expected, the truncated βDG fragment failed to bind to the dystrophin WW-ZZ domain.

  Binding of βDG to both plectin fragments was effi ciently blocked by the dystrophin WW-ZZ domain (Fig. 9 E). When dystrophin was added to the overlay solutions at an equimolar ratio, βDG–plectin binding was only slightly reduced, but

  Figure 5. Distribution of plectin isoforms 1 and 1f in muscle during the course of MD in mdx mice. Cross sections of quadriceps from 4- and 14-wk-old mdx and normal control mice were immunolabeled with plectin 1f– (A–C and G–I) and plectin 1–specifi c antibodies (D–F, J, and K). Secondary antibodies used were Cy5 labeled. Areas of unaffected (B and E), actively regenerating (C and F), and regenerated (H, I, and K) mdx muscles are shown. Insets in G–I show autofl uorescence after excitation at 488 nm, with autofl uorescent fi bers corresponding to type 2A fi bers. n, sectioned nuclei. Bar, 20 μm.

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  Figure 6. Comparative (semiquantitative) IB analysis of total cell lysates and microsome fractions from control and mdx skeletal muscles. (A and B) Scans of typical bands obtained by IB. (C and D) Bands were evaluated as described in Fig. 3. Signals were normalized to tubulin (total lysates) or total pro- tein content (microsome fractions) and are shown relative to protein amounts in control (wild type) samples (100%). Values represent means ± SD (error bars) of at least three gel runs using samples from two independent preparations.

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  increasing the molar ratios to 1:5 or 1:10 in favor of dystrophin were observed in two additional lanes (Ple E1–12 and Utr ABD; led to strongly reduced binding and no binding, respectively. Fig. 9 E), suggesting that the dystrophin WW-ZZ domain me- Unexpectedly, in this competition experiment, positive signals diated the indirect binding of βDG to the ABDs of utrophin

  

  Figure 7. Expression of DG and utrophin during the course of MD in mdx mice and localization of DG in teased muscle fi bers. (A–L) Cross sections of gastrocnemius from 2-, 4-, and 14-wk-old normal control (A, B, E, F, I, and J) and mdx (C, D,

  G, H, K, and L) mice were immunolabeled using antibodies specifi c for βDG (A, C, E, G, I, and K) and utrophin (B, D, F, H, J, and L). Note the expression (albeit reduced at ages 2 and 14 wk) of βDG in mdx muscle fi bers. Utrophin is present at the sarcolemma of muscle fi bers from young (<4 wk old) animals (normal and mdx) as well as in regenerating fi bers from adolescent and adult mdx animals; at these stages, utrophin localization in nonregenerating fi bers is virtually con- fi ned to myotendinous junctions both in normal and mdx mice (arrowheads in F and H). Asterisks in H and L denote areas of active regeneration in mdx muscle. (M–R) Teased fi bers from normal and mdx muscles from 8-wk-old mice were stained with pan-plectin mouse antiserum #123 (pan), rabbit antiserum to plectin 1f (insets) and rabbit antiserum #1710 (AS), or mouse mAbs (insets) to βDG. Control and mdx samples were stained in parallel, and images were recorded using the same settings. Q and R show magnifi cations of the areas labeled Q and R in the insets in N and P. Bars (A–L), 50 μm; (M–P) 20 μm. REZNICZEK ET AL. 971 JCB • VOLUME 176 • NUMBER 7 • 2007 972

  and plectin. To confi rm the simultaneous binding of WW-ZZ domains to plectin and βDG, we performed an overlay assay in which the utrophin WW-ZZ domain was immobilized on microtiter plates and incubated with constant amounts of Eu 3+ - labeled βDG in the presence of increasing concentrations of labeled or unlabeled versions of the plectin ABD (Fig. 9 F). In the fi rst case, the amounts of labeled protein bound remained unchanged (Fig. 9 F, gray bars), whereas signals were additive (Fig. 9 F, black bars) in the latter, clearly demonstrating that plectin and βDG bound to independent binding sites within the WW-ZZ domain.

  Muscle formation is an incremental process in which differenti- ating myoblasts fuse and form primary and fi nally secondary myotubes. As this process involves massive rearrangements of the cytoskeleton, it was not unexpected to fi nd that plectin iso- forms were differentially expressed during its course. Of special interest was the striking similarity of the temporal expression patterns of plectin 1f and dystrophin, which suggested a role for this particular plectin isoform in the formation and maturation of costameres. This is supported by the observed exclusive membrane association of a recombinant version of this isoform in differentiated myotubes but not at the myoblast stage, where dystrophin is absent. A similar role of plectin had been sug- gested previously by Schröder et al. (2000, 2002), who con- cluded from their myoblast differentiation experiments that the association of plectin with Z-disks is a prerequisite for formation of the intermyofi brillar desmin cytoskeleton and, furthermore, that plectin is a component of primary longitudinal adhe- sion structures, which are precursors of costameres that form mature costameres only after being subjected to contractile forces. This would also explain the apparent discrepancy in plectin 1f localization in the tissue and in teased fi bers (sarco- lemma and Z-disks) versus transfected myotubes (sarcolemma only), as the latter represent a less mature stage. Interestingly, using mAb 121 to plectin, Schröder et al. (2002) identifi ed a membrane-associated plectin variant that is up-regulated during human myotube differentiation. Our results would suggest that this variant is plectin 1f. However, it is unexplainable how a mAb with an epitope in plectin’s rod domain could specifi cally detect one rod-containing isoform (1f) over others.

  Immunostaining of muscle tissue revealed that plectin ex- pression levels in individual fi bers varied and were dependent on the fi ber type. In cross sections of normal striated human muscle, a moderate to intense cytoplasmic and sarcolemmal staining of plectin has been reported in type 1 (slow twitch) fi bers, whereas only faint staining of the sarcolemma was ob- served in type 2 (fast twitch) fi bers (Schröder et al., 1997). In the present study, we show using an antiserum to plectin not discriminating among isoforms that in quadriceps (a typical fast muscle composed of mainly type 2 fi bers), plectin clearly was localized at Z-disks in both type 2A and 2B fi bers, with a stronger signal in 2A fi bers. This corresponds well with the in- tense staining obtained with plectin 1f–specifi c antibodies in this fi ber type. Neither with anti–plectin 1f nor anti–plectin 1 antibodies did we detect substantial Z-disk staining in 2B fi bers. Based on this observation, one other plectin isoform ex- pressed in skeletal muscle, plectin 1b or 1d, must be associated

  Figure 8. Co-IP and ultrastructural colocalization of plectin and DG. (A) Cell lysates from C2C12 myoblasts (M), mouse keratinocytes (K), and CaCo-2 (C) cells were subjected to IP using anti-βDG antiserum; antibodies used for detection are indicated. (B) mdx muscle lysates were immunoprecipitated with the indicated antibodies and probed for βDG. (C–E) Preembedding immunogold labeling of teased muscle fi bers extracted with Triton X-100. Gold particles labeling plectin (5 nm) and βDG (10 nm) were silver enhanced. In C, note the intense labeling of both proteins at the plasma membrane lo- cally separated from the muscle fi ber. The exterior of the fi ber (e) contains extracellular material, whereas the blistered interior (i) is empty. In D, a sarcomere positioned beneath the sarcolemma is shown. Although the plasma membrane is lost by Triton X-100 extraction, βDG as well as plectin remain anchored to fi lamentous structures (arrowheads) in the subsarcolemmal region. Also note plectin labeling in the interior of the fi ber at Z-disks. In E, details of a sarcomeric region proximal to a Z-disk are shown. Labeling of βDG is restricted to the subsarcolemmal region with incomplete detachment of the sarcolemma. While most of the βDG is located at fi lamentous structures (white arrowheads), it can also be observed (arrow) in association with the sarcolemma (black arrowheads). Bars, 500 nm.

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Discussion Distinct regulation and localization of plectin isoforms in skeletal muscle

  with Z-disks in type 2B fi bers. From our overexpression experi- ments, we conclude that this isoform is plectin 1d, as it was found localized exclusively at Z-disks. At this time, the molecular mechanism of this targeting is unknown.

  Plectin is associated with the DGC via multiple interfaces

  Using co-IP and in vitro binding assays, we demonstrate direct interactions of plectin via multiple interfaces with components of the DGC, including (1) direct binding of its plakin domain to the cytoplasmic domain of βDG; (2) direct binding of its C-terminal portion to βDG; (3) binding of its ABD to the WW-ZZ domains of dystrophin and utrophin; and (4) binding of its ABD to the ABD of utrophin (Figs. 4 and 9, schematics). Whether the ABD of plectin would also interact with that of dystrophin re- mains an open question considering that the functionalities of the ABDs of dystrophin and utrophin differ (Rybakova et al., 2006), whereas their WW-ZZ domains are highly conserved (Hnia et al., 2007). Plectin and dystrophin had previously been coimmunoprecipitated from muscle lysates, but their interaction was assumed to be indirect via actin (Hijikata et al., 2003). In dystrophin-lacking mdx mice, one may thus expect to fi nd the reduced sarcolemma association of plectin, but our immuno- fl uorescence and tissue fractionation experiments revealed that plectin was instead enriched at the sarcolemma of mdx muscle. It was widely believed that dystrophin defi ciency in skeletal mus- cles of mdx mice and DMD patients leads to the reduced expression and sarcolemmal association of dystrophin-associated proteins, including a strong reduction or even absence of βDG immuno- reactivity (Ohlendieck and Campbell, 1991) despite its mRNA levels being similar to those in normal samples (Ibraghimov- Beskrovnaya et al., 1992; Rouger et al., 2002). However, in a recent study, Cluchague et al. (2004) challenged this view when they demonstrated that in mdx muscle samples treated with 2% cholate, βDG was detectable at levels comparable with those of wild-type samples. The authors proposed that βDG was targeted to the plasma membrane normally in dystrophin- defi cient mdx muscles but remained inaccessible to antibodies and, when tissues were lysed, became part of an SDS-insoluble pool. Using our protocols, we also found considerable levels of βDG in mdx skeletal muscle microsome fractions ( 50% of wild type even without cholate treatment), and we were able to immunodetect βDG with variable intensities throughout mdx muscle regeneration. Thus, our results support the hypothesis of Cluchague et al. (2004), and the direct interaction of plectin with βDG provides an explanation for the observed increase in sarcolemmal plectin in mdx muscle (Fig. 10). In the absence of dystrophin, more plectin can bind to βDG, causing at the same time the redistribution and accumulation of βDG above Z-disks, where plectin is normally localized. Matching our βDG immuno- staining results, Yurchenco et al. (2004) have observed a cor- responding redistribution of αDG in teased mdx fi bers.

  It has been suggested that utrophin could substitute for dystrophin in dystrophic muscles, but its intimate association with βDG may be limited to the time of regeneration only (Tinsley et al., 1998). The 50% reduction of utrophin observed in

  Figure 9. Direct binding of DG to N- and C-terminal domains of plectin. (A) Schematic representation of plectin, β DG, dystrophin, and protein fragments used for in vitro inter- action assays. Binding interfaces between plectin, dystrophin, and βDG are indicated by brackets connected by lines. ABD, actin-binding domain; IFBD, IF-binding domain; TM, trans- membrane domain; DBS, dystrophin-binding site. N and C termini of proteins are indicated. (B–E) Blot overlay assay. A panel of protein fragments recombinantly expressed in bacteria (B; Coomassie) was immobilized on nitrocellulose membranes and overlaid with βDGcyt (C) or βDGcyt∆DBS, a shorter fragment comprising amino acids 765–857 and lacking the dystrophin-binding site (D). Bound proteins were detected using mAbs to βDG (C) or via the S tag contained in β DGcyt∆DBS (D). In E, membranes were overlaid with β DGcyt in the presence of Dys WW-ZZ, a fragment corre- sponding to the WW-ZZ domain of dystrophin. Molar ratios (βDGcyt/Dys WW-ZZ) were 1:1, 1:5, and 1:10 in the large, small top, and small bottom panels, respectively. (F) Microtiter plate competition binding assay. Recombinantly expressed and purifi ed WW-ZZ domains of utrophin (immobilized) were overlaid with constant amounts of a Eu 3+ -labeled version of the full-length cytoplasmic domain of βDG (βDGcyt; white bars; 100% indicated by the dotted line) and increasing amounts of unlabeled (gray bars) or Eu 3+ -labeled (black bars) recombinant plectin ABD. Data represent mean ± SD (error bars) of a typical experiment performed with triplicate wells.

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  • REZNICZEK ET AL. 973
JCB • VOLUME 176 • NUMBER 7 • 2007 974

  microsome preparations from muscle of 10-wk-old mdx mice despite the overall higher expression of utrophin ( 150% of wild-type levels) would support this notion. Furthermore, this observation also suggests that plectin binds to βDG with a higher affi nity than utrophin. Remaining sarcolemmal utrophin staining that was observed in transgenic mdx mice overexpressing Dp71, a short splice variant of dystrophin lacking rod and N-terminal domains (Cox et al., 1994; Greenberg et al., 1994), was explained by Cox et al. (1994) to possibly be caused by the binding of utro- phin to subsarcolemmal actin via its ABD. Our fi nding that βDG immunostaining signals observed in mdx tissue from 4-wk-old animals was almost as strong as in normal muscle would also fi t the proposed model (Fig. 10), as the higher expression of utro- phin during the phase of peak necrosis/regeneration may dis- place plectin from βDG and, thus, restore accessibility of the epitopes masked by plectin. Similarly, the overexpression of Dp71 (or other dystrophin or utrophin deletion variants) in mdx restored normal DGC components, whereas a full phenotypic rescue was achieved only by proteins with functional ABDs and intact C-terminal βDG-binding domains (Tinsley et al., 1998).

  Plectin acts as a universal mediator of IF anchorage

  The DGC has been considered to be responsible for connecting the subsarcolemmal actin cytoskeleton to the ECM, and disrup- tion of this link causes a dystrophic phenotype. However, in re- cent years, it has been established that the contractile actions of a muscle fi ber are mechanically integrated by desmin IFs, which are responsible for linking individual myofi brils laterally with each other and to the sarcolemma at the level of the Z-disks. Previous studies have implicated the DGC as the transmem- brane complex linking the IF network with the ECM (for r eviews see Blake and Martin-Rendon, 2002; Capetanaki, 2002; Paulin and Li, 2004). The necessary link would be created by an α-dystrobrevin–synemin/syncoilin–desmin bridge. Synemin may also directly interact with vinculin, providing an alternative anchorage of desmin IFs to costameres (Bellin et al., 2001). Plectin directly interacts with desmin via its C-terminal IF- binding domain (Reipert et al., 1999) and also with multiple components of the DGC, including its transmembrane core pro- tein βDG. Thus, we propose that plectin acts as a direct linker between the DGC and the desmin IF network. There is prece- dence for such a function of plectin in basal keratinocytes, where the protein directly links the keratin IF network to the cyto- plasmic domain of the β subunit of the laminin receptor integrin α6β4 (Rezniczek et al., 1998). It could be that synemin plays the essential role in establishing the direct linkages between heteropolymeric IFs and the myofi brillar Z-disk and costameric regions, and plectin might only provide additional structural support at these sites. However, this would be in confl ict with observations in differentiating human skeletal muscle cultures, where plectin was already localized in a cross-striated pattern, whereas desmin was still found in longitudinal fi laments (Schröder et al., 2000).

  Recently, it was shown that besides type III and IV IFs, the cytokeratins K8 and K19 are also expressed in striated mus- cle and localize to Z-disks and M-lines and that K19 directly interacts with the dystrophin ABD (Stone et al., 2005). Whereas plectin has been shown to directly bind to keratins 5, 14, and 18 (Geerts et al., 1999; Steinböck et al., 2000), it is unknown whether it can also interact with the muscle-specifi c keratins and possibly plays a role in their anchorage as well.

  Based on our observations, we propose the following model for plectin’s association with the DGC (Fig. 10). Because (1) binding of plectin to βDG via its C-terminal binding site was abolished in the absence of the C-terminal part of βDG’s cytoplasmic domain (harboring the PPxY motif required for interaction with the WW domain of dystrophin and utrophin; see Ilsley et al., 2002 for a d

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